Structural insights into the human PA28-20S proteasome enabled by efficient tagging and purification of endogenous proteins.

ABSTRACT: The ability to produce folded and functional proteins is a necessity for structural biology and many other biological sciences. This task is particularly challenging for numerous biomedically important targets in human cells, including membrane proteins and large macromolecular assemblies, hampering mechanistic studies and drug development efforts. Here we describe a method combining CRISPR-Cas gene editing and fluorescence-activated cell sorting to rapidly tag and purify endogenous proteins in HEK cells for structural characterization. We applied this approach to study the human proteasome from HEK cells and rapidly determined cryogenic electron microscopy structures of major proteasomal complexes, including a high-resolution structure of intact human PA28αβ-20S. Our structures reveal tha

SUBMITTER: Zhao J 

PROVIDER: S-EPMC9388094 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature